GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of Kallman patients. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, three articles were published: 1) We previously showed that CCDC141 knockdown leads to impaired migration of GnRH neurons but not of olfactory receptor neurons. In this collaboration, the phenotype and prevalence of CCDC141 mutations in patients with idiopathic hypogonadotropic hypogonadism/Kallman Syndrome (IHH/KS) is further described. Using autozygosity mapping, candidate gene screening, whole-exome sequencing, and Sanger sequencing, those individuals carrying deleterious CDCD141 variants and their phenotypes were determined in a cohort of 120 KS families. The studies revealed nine affected individuals from four independent families in which IHH/KS is associated with inactivating CCDC141 variants, revealing a prevalence of 3.3%. Affected individuals (with the exception of one family who concomitantly have FEZF1 mutations) have normal olfactory function and anatomically normal olfactory bulbs. Four affected individuals show evidence of clinical reversibility. In three of the families, there was at least one more potentially deleterious variant in other known puberty genes with evidence of allelic heterogeneity within respective pedigrees. These studies confirm that inactivating CCDC141 variants cause normosmic IHH but not KS. This is consistent with our previous in vitro experiments showing exclusively impaired embryonic migration of GnRH neurons upon CCDC141 knockdown. These studies expand the clinical and genetic spectrum of IHH and also attest to the complexity of phenotype and genotype in IHH. 2) Fertility relies on the proper functioning of the hypothalamicpituitarygonadal axis. The hormonal cascade begins with hypothalamic neurons secreting gonadotropin releasing hormone (GnRH) into the hypophyseal portal system. In turn, the GnRH activated gonadotrophs in the anterior pituitary release gonadotropins, which then act on the gonads to regulate gametogenesis and sex steroidogenesis. Finally, sex steroids close this axis by feeding back to the hypothalamus. Despite this seeming straightforwardness, the axis is orchestrated by a complex neuronal network in the central nervous system. For reproductive success, GnRH neurons, the final output of this network, must integrate and translate a wide range of cues, both environmental and physiological, to the gonadotrophs viapulsatile GnRH secretion. This secretory profile is critical for gonadotropic function, yet the mechanisms underlying these pulses remain unknown. Literature supports both intrinsically and extrinsically driven GnRH neuronal activity. However, the caveat of the techniques supporting either one of the two hypotheses is the gap between events recorded at a single-cell level and GnRH secretion measured at the population level. This review compiles data about GnRH neuronal activity focusing on the physiological output, GnRH secretion. 3) Tissue autofluorescence occurs as components, such as red blood cells, naturally fluoresce across multiple wavelengths, and this can be enhanced by the fixation process. Auto-fluorescence is problematic because it can mask or interfere with the fluorescence of experimental reporters such as antibodies, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence. Therefore, one must reduce autofluorescence to accurately analyze fluorescent reporters. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins - making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher (Biotium). The protocols described in this unit demonstrate how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identified optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy.